Fig 1.
Sample collection site, clinical and pathological observations in infected goats, and the identification of cpCoV.
The sample collection sites are marked by dots (The base layer link of the map is from https://vgimap.tianditu.gov.cn/ according to GS(2024)0568)) (A). Diseased goats under 2 months of age showed clinical signs, including depression symptoms, varying degrees of diarrhea, weight loss (B), and intestinal tract bleeding (C). IFA detection of cpCoV-infected HRT-18G (D), MDBK, (E) and HT-29 (F) cells by anti-Spike protein monoclonal antibody. Replication of cpCoV/AHFY2302G in HRT-18G, HT-29 and MDBK cells. Cells were infected with isolate AHFY2302G and harvested at 0-36 hpi, and replication was measured by qRT-PCR (G). Transmission electron micrograph of cpCoVs using negative staining with 2% sodium phosphotungstate. Coronaviruses were observed as typical corona-shaped particles of 70–90 nm in diameter (H).
Table 1.
Detection and isolation of caprine coronavirus in goat samples from different provinces/autonomous regions of China.
Fig 2.
(A) Genomic organization of cpCoV and other β-CoV strains in the region between the Spike (S) gene and the NS5a gene.
Distinct open reading frame (ORF) patterns were detected in β-CoV 1 in this region. Black triangles indicate stop codons. Horizontal dotted lines indicate a deletion. (B) Nuclear acid comparison of Embecovirus ORF1ab and structural proteins. CpCoVs were used as reference. Numbers and colors in the heat map represent the percentage identities between selected strains and cpCoVs. Pairwise identity scores were generated using MegAlign software, and the heat map was generated in Prism software. Heatmap of homology analysis based on the complete genome (C), and the S (D) and NS4a-4b (E) genes of cpCoV and reference strains.
Fig 3.
Phylogenetic tree based on the complete genome
(A), S gene (B), RNA-dependent RNA polymerase (RdRp) (C), and N gene (1,347 nt) (D) of cpCoV and representative CoVs. The alignment of each gene was manually trimmed to 31,005 nucleotides (nt) for the whole genome, 4,092 nt for the S gene, 2,783 nt for RdRp, and 1,347 nt for the N gene. Analyses were conducted using MEGA software version 6.0 (http://www.megasoftware.net) with the neighbor-joining algorithm. Bootstrap values (indicated on each branch) were calculated with 1,000 replicates. Circles indicate the cpCoV strains. Scale bar = nucleotide substitutions per site. CoV, coronavirus; PDCoV, porcine deltacoronavirus; PEDV, porcine epidemic diarrhea virus; PHEV, porcine hemagglutinating encephalomyelitis virus; PRCV, porcine respiratory coronavirus; SARS, severe acute respiratory syndrome; TGEV, transmissible gastroenteritis virus. (E) Recombination analysis of the genomes of cpCoV/AHFY2302G, DcCoV-HKU23, canine CoV D200NS_THA_2022, and K39. Bootscan analysis was performed with Simplot, version 3.5.1.
Fig 4.
Pathogenicity experiments in cpCoV-infected goats.
Goats were intranasally inoculated with 5 × 105 TCID50 virus. Diarrhea (B) and nasal discharge (D) were observed in goats in the challenge groups (CC-Goat), whereas the control group (NC-Goat) goats were normal (A and C). (E) Diarrhea index was evaluated in the CC-Goat and NC-Goat groups; the goats in the CC-Goat group showed apparent diarrhea. CpCoV viral RNA shedding was detected in rectal (F), nasal (G), and throat (H) swabs. The viral load was detected in almost all organs of goats (I). Histopathological examination of cpCoV-infected goats. The alveolar wall of the CC-Goat group was significantly thickened, epithelial cells were shed, and the alveolar cavity was filled with lymphocytes (J and K). The duodenum (L and M), ileum (N and O), cecum (P and Q), and rectum (R and S) had different degrees of villus atrophy and severe mucosa abscission. Lymphocytes in mesenteric lymph nodes were markedly reduced and sparse (T and U).
Fig 5.
Pathogenicity experiments in cpCoV-infected calves.
The animals were intranasally inoculated with 2 × 106 TCID50 virus. The diarrhea index was evaluated in the four groups. Calves in the CC-7 and CC-14 groups showed apparent diarrhea (A) and an elevated temperature (B) compared with the NC groups. CpCoV viral RNA shedding was detected in rectal (C), nasal (D), and throat (E) swabs. Viral load was detected in almost all organs of calves in CC-7 and CC-14 groups (F). Histopathological examination of cpCoV-infected calves. Pathological changes in calves in the CC-7 group were mainly in the intestine, with severe disintegration and shedding of the intestinal villi in the duodenum (G and H), ileum (I and J), jejunum (K and L), cecum (M and N), colon (O and P), and rectum (Q and R).